Antiviral salts

ABSTRACT

This invention relates to antiviral agents, in particular to salts useful in the treatment of infections caused by Picornaviridae, such as human rhinovirus (HRV) and methods for their preparation. The invention also relates to the use of these salts in the treatment of picornavirus infections. The salts of this invention are especially suitable for use in the treatment of HRV, however it is to be understood that the invention is also applicable to other viruses of the picornavirus family.

FIELD OF THE INVENTION

This invention relates to antiviral agents, in particular to saltsuseful in the treatment of infections caused by Picornaviridae, such ashuman rhinovirus (HRV) and methods for their preparation. The inventionalso relates to the use of these salts in the treatment of picornavirusinfections. The salts of this invention are especially suitable for usein the treatment of HRV, however it is to be understood that theinvention is also applicable to other viruses of the picornavirusfamily.

BACKGROUND OF THE INVENTION

Human rhinoviruses (HRV) are particular picornaviruses and are the mostcommon infectious viral agents of the respiratory system. Indeed, theyare the primary cause of the “common cold”. The virus particles consistof a modular protein shell (the capsid) which encloses a short singlestrand of RNA.

One particular anti-picornavirus compound, which functions as acapsid-binding inhibitor and has been the subject of recent humanclinical trials, is4-[2-[1-(6-methyl-3-pyridazinyl)-4-piperidinyl]-ethoxy]benzoate—otherwiseknown as “Pirodavir”. Pirodavir, being lipophilic, displays poor aqueoussolubility and is often formulated with a cyclodextrin host. Theresultant complex is more soluble in aqueous media, and allows deliveryof the drug through such means as a nasal spray. It has, however, beenshown that facile endogenous cleavage of the ester moiety in pirodavirleads to poor bioavailability of the molecule regardless of the chosenexcipient. Furthermore, and in addition to cost, cyclodextrin hostmolecules have been shown to be otherwise disadvantageous in theformulation of pharmaceuticals (Int. J. Pharm., 2002, 246, 25-35).Another promising anti-picornavirus compound, “Pleconaril”, has beenshown to interact adversely with patients using oral contraceptiveformulations.

Clearly there is a need for an anti-picornavirus compound which displaysdesirable aqueous solubility properties, is stable under physiologicalconditions and has few adverse interactions. Additionally it isdesirable that such a compound would be relatively easy to form andwould be stable on storage in solid and solution form.

SUMMARY OF THE INVENTION

W02002/50045 discloses a novel class of antiviral compounds that werefound to exhibit particularly favourable anti-picornoviral properties.It has now been surprisingly discovered that two particular acidaddition salts, namely the phosphate and sulfate, of compound 35 ofTable 3 of W02002/50045, offer particular advantage over other compoundscurrently proposed for the treatment of picornarviruses. Compound 35(the ‘free base’—represented by formula I below) has been shown todisplay potent activity against both HRV2 (IC₅₀ 0.001 [1.g/mL) and HRV14(IC₅₀ 0.005 ug/mL). These tests were conducted using dimethylsulfoxide(DMSO) as the carrier solvent. The chemical name given to the compoundof formula I is6-{2-[1-(6-methyl-3-pyridazinyl)-4-piperidinyl]ethoxy}-3-ethoxy-1,2-benzisoxazole.

Particularly preferred stoichiometric forms of the acid addition saltsof the free base (formula I) in accordance with the invention are:

-   -   a) the acid addition salt of phosphoric acid (2 equivalents)        with the free base; and    -   b) the acid addition salt of sulfuric acid (1 equivalent) with        the free base.

Accordingly in one aspect the invention provides the phosphoric acid (2eq) addition salt of the free base of the compound of formula I.

In another aspect the invention provides the sulfuric acid (1 eq)addition salt of the free base of the compound of formula I.

These salts advantageously have the following properties:

-   -   a) they display similar potent biological activity to the free        base (for example EC₅₀ vs human rhinovirus is 14.4 nM for free        base of compound of formula I and is 17.7 nM for phosphoric acid        (2 eq) addition salt of free base of compound of formula I);    -   b) they have substantially greater aqueous solubility over other        salts of the free base as well as the free base itself;    -   c) they form readily and are easy to isolate; and    -   d) they crystallise readily, and those crystalline salts are        stable both in the mother liquor and in isolated form.

These properties are recognised as being particularly desirable for asalt of the free base of a biologically active molecule to have, toassist with handling, formulation, etc.

In another aspect the invention provides pharmaceutical compositionscomprising either or both of the salts and one or more carriers.

In another aspect the invention provides a method of forming thephosphoric acid addition salt comprising the step of reacting the freebase with phosphoric acid. Preferably the free base is contacted withapproximately 2 molar equivalents of phosphoric acid, more preferablybetween about 1.8 and 2.2 molar equivalents of phosphoric acid.

In another aspect the invention provides a method of forming thesulfuric acid addition salt comprising the step of reacting the freebase with sulfuric acid. Preferably the free base is contacted withapproximately 1 molar equivalent of sulfuric acid, more preferablybetween about 0.8 and 1.2 molar equivalents of sulfuric acid.

In another aspect the present invention provides a method of treating apicornavirus infection in a subject in need thereof comprisingadministering either or both of the salts.

In another aspect the present invention provides a method of preventinga picornavirus infection in a subject in need thereof comprisingadministering either or both of the salts.

In another aspect the present invention provides the use of either orboth of the salts in the manufacture of a medicament for the treatmentof a picornavirus infection in a subject in need thereof.

In another aspect the present invention provides the use of either orboth of the salts in the manufacture of a medicament for the preventionof a picornavirus infection in a subject in need thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is an image obtained using Scanning Electron Microscopy (SEM),with scale, of crystals of the phosphoric acid (2 eq) addition salt ofthe compound of formula I in accordance with a preferred embodiment ofthe invention.

FIG. 2 is an X-Ray Powder Diffraction spectrum of the phosphoric acid (2eq) addition salt of the compound of formula I in accordance with apreferred embodiment of the invention. The calculated 2θ peak values(±0.2°) are 3.3°, 6.7°, 12.8°, 13.3°, 14.1°, 19.2°, 20.0°, 21.1° and22.4°.

FIG. 3 is an X-Ray Powder Diffraction spectrum of the sulfuric acid (1eq) addition salt of the compound of formula I in accordance with apreferred embodiment of the invention. The calculated 2θ peak values(±0.2°) are 3.3°, 6.7°, 13.2°, 15.5°, 15.9°, 18.9° and 22.9°.

FIG. 4 shows the water content results obtained for trial 1 and trial 2as described in Tables 3 and 4 for the bis-dihydrogenphosphate salt inaccordance with a preferred embodiment of the invention.

FIG. 5 shows the anhydrous assay results obtained for trial 1 and trial2 as described in Tables 3 and 4 for the bis-dihydrogenphosphate salt inaccordance with a preferred embodiment of the invention.

FIG. 6 shows the total related substance results obtained for trial 1and trial 2 as described in Tables 3 and 4 for thebis-dihydrogenphosphate salt in accordance with a preferred embodimentof the invention.

FIG. 7 is an x-ray crystal structure of the monosulfate salt inaccordance with a preferred embodiment of the invention (a) shows anasymmetric unit (ORTEP drawing showing 30% probability ellipsoids) and(b) shows packing down the b-axis.

FIG. 8 is an x-ray crystal structure of the bis-dihydrogenphosphate saltin accordance with a preferred embodiment of the invention (a) shows anasymmetric unit showing the H₂PO₄ and H₂O disorder (ORTEP drawingshowing 30% probability ellipsoids) (b) shows an asymmetric unit removedH₂O molecules and H₂PO₄ disorder (ORTEP drawing showing 30% probabilityellipsoids) (c) shows packing down the b-axis.

FIG. 9 shows a typical HPLC chromatogram for the bis-dihydrogenphosphatesalt in accordance with a preferred embodiment of the invention. It maybe assumed that the salt will dissociate, at least to some degree,during analysis under these conditions.

DETAILED DESCRIPTION OF THE INVENTION

Without wishing to be bound by theory, it is believed that of thenitrogen atoms present within the compound of formula I, only thepiperidinyl nitrogen atom is readily protonated by an acid.

The other two basic nitrogen atom containing moieties within themolecule (being the benzisoxazolyl and pyridazinyl) are relatively weakbases, and it was believed that only reaction of either of these groupswith a strong acid would produce a stable acid addition salt. On theother hand, it was also believed that the free base compound issensitive to the action of strong acids, which presented anotherchallenge to increasing the aqueous solubility of the free base—beingachieving a balance between stable salt formation and degradation of themolecule.

It has now been surprisingly discovered that the reaction of the freebase (formula I) with approximately 1 molar equivalent of sulfuric acidproduces the monosulfate salt of the bis-protonated free base (formulaII) remarkably without any observed degradation of the molecule. As usedherein this addition salt is referred to as6-{2-[1-(6-methyl-3-pyridazinyl)-4-piperidinyl]ethoxy}-3-ethoxy-1,2-benzisoxazolesulfate salt (1:1). The term “(1:1)” denotes the molar ratio of thebis-protonated conjugate acid of the free base of formula I to sulfatedi-anion.

Furthermore, it has been surprisingly discovered that the reaction ofthe free base (formula I) with approximately 2 molar equivalents ofphosphoric acid produces the bis-dihydrogenphosphate salt of thebis-protonated free base (formula III), again without any observeddegradation. As used herein this addition salt is referred to as6-{2-[1-(6-methyl-3-pyridazinyl)-4-piperidinyl]ethoxy}-3-ethoxy-1,2-benzisoxazolebis-dihydrogenphosphate salt (1:2). The term “(1:2)” denotes the molarratio of the bis-protonated conjugate acid of the free base of formula Ito dihydrogenphosphate anion.

Whilst it may be the case that a given salt of a given active compoundmight display one or more properties which make it suitable for use as apharmaceutical, what is particularly surprising about the sincediscovered properties of these two salts is that they substantiallyoutperform other salts of the compound of formula I.

Of the reaction products formed from reaction of the free base offormula I with the following acids: acetic acid; citric acid; benzoicacid; fumaric acid; D-gluconic acid; hydrobromic acid; hydrochloricacid; maleic acid; nitric acid; oxalic acid; phosphoric acid; sulfuricacid; L-(+)-tartaric acid; toluenesulfonic acid; and methanesulfonicacid, only the salts formed from: nitric acid; phosphoric acid; sulfuricacid; hydrochloric acid; oxalic acid; and L-(+)-tartaric acid weredemonstrated to produce salts or co-crystals of the free base whichwould furthermore potentially be suitable for pharmaceutical use.

The acid addition salt formed from the reaction of nitric acid with thefree base of formula I displayed sub-optimal aqueous solubility. Theproduct formed from contacting the free base of formula I with tartaricacid was subsequently shown to be a substantially co-crystallinecompound, and not the desired acid addition salt.

The following techniques were used to characterise the acid additionsalts and determine their solubility and stability:

-   -   a) Visual inspection;    -   b) Raman spectroscopy (to determine salt/co-crystal formation);    -   c) X-Ray Powder Diffraction spectroscopy (to determine the        crystallinity of the product);    -   d) High Performance Liquid Chromatography (HPLC) analysis        (reverse phase; to determine by-product formation);    -   e) Differential Scanning calorimetry (to determine melting        point/decomposition temperature);    -   f) Elemental analysis (to determine purity/hygroscopicity);    -   g) Solubility determination (wherein 10 mg of the salt was        contacted with a limited quantity of water for a time, and under        conditions, such that solid necessarily remained and could be        filtered off before the concentration of the saturated solution        could be determined using HPLC);    -   h) Four week storage under different stress conditions (25° C.        dry, 25° C. with 75% relative humidity (rh), 25° C. with 90% rh,        40° C. dry, 40° C. with 75% rh, 40° C. with 90% rh) including        non-invasive characterisation by Near Infrared spectroscopy and        Raman spectroscopy during the test as well as HPLC analysis at        completion of the test.

Further details of the characterisation methods and parameters areprovided in Example 5.

Table 1 shows comparative results which have been obtained for the acidaddition salts of the free base of formula I. The darkened cellsindicate results which disfavour the use of that salt as apharmaceutical. As can be seen, salts 2 and 4 significantly outperformthe other salts with respect to aqueous solubility and storagestability. The results of elemental analysis of the 2:1 oxalic acid:freebase salt were not in agreement with the predicted results, andaccordingly the slightly higher solubility of 1873 mg/mL was discountedas a meaningful result.

TABLE 1 Elemental Analysis Acid by- (in agreement (stoichiometryproduct? with calculated Salt base:acid) (HPLC) composition) SolubilityStability 1 HCl (1:1) No Yes  835 μg/mL isolated salt: slowmorphological change at 25° C./90% rh (pH 1.93) isolated salt: fastmorphological change at 40° C./90% rh 2 H₂SO₄ (1:1) No Yes 1348 μg/mL nodegradation/change under all conditions (pH 1.99) not hygroscopic 3H₃PO₄ (1:1) No Yes  462 μg/mL no degradation/change under all conditions(pH 2.12) not hygroscopic 4 H₃PO₄ (1:2) No Yes 1384 μg/mL nodegradation/change under all conditions (pH 1.78) not hygroscopic 5HO₂CCO₂H (1:1) Yes Yes  625 μg/mL isolated salt: morphological change at25° C./90% rh (pH 2.00) not hygroscopic 6 HO₂CCO₂H (1:2) Yes No 1873μg/mL hygroscopic under conditions of 75% rh and 90% rh (pH 1.94)

Without wishing to be bound by theory, whilst two of the three basicnitrogen containing moieties on the compound of formula I are relativelyweak it would appear that their participation in salt formation does notpredicate the use of a necessarily strong acid. Phosphoric acid is aweaker acid than other inorganic acids such as sulfuric, hydrochloricand nitric acid. Nonetheless, and quite surprisingly, the phosphoricacid addition salt (salt 4 of Table 1) has been demonstrated to be notonly crystalline, but also stable under a range of conditions. In fact,as can be seen in Table 1, the stability of the salt formed withhydrochloric acid (salt 1 of Table 1) was inferior to the salt formedwith phosphoric acid (salt 4 of Table 1).

An additionally surprising result is that double protonation of the freebase affords a product (salts of both formulae II and III) whichdisplays greater stability than the product of single protonation of thefree base by hydrochloric acid.

Furthermore, it would appear that the solubility of thebis-dihydrogenphosphate salt of formula III is greater than would havebeen predicted from extrapolation of the solubility of themono-dihydrogenphosphate salt (salt 3 of Table 1).

The stability of the bis-dihydrogenphosphate salt has been tested usingthe test parameters of

-   -   i) Appearance    -   ii) Water content    -   iii) Assay (Anhydrous basis)    -   iv) Related Compounds (Reported as Maximum Individual)    -   v) Related Compounds (Total)

The specifications for the bis-dihydrogenphosphate salt are shown inTable 2. The characterisation methods, parameters and exemplary HPLCassay data are provided in Example 5.

TABLE 2 Bis-dihydrogenphosphate salt specifications TestSpecification 1. Appearance Yellow free flowing powder. 2.Identification The infrared spectrum of the sample exhibits (InfraredSpectrum) maxima at the same wavelengths as that of a similarpreparation of the reference standard. 3. HPLC Assay 95.0% to 105.0%(anhydrous, solvent free). 4. Related Substances i. Single maximumimpurity should not be (HPLC) more than 0.5%. ii. Total impuritiesshould not exceed 1.0%. 5. Water Content Not more than 5.0%. (KarlFischer) 6. Residual Solvents by GC Isopropanol: Not more than 0.5%Acetone: Not more than 0.5%

Two batch samples of the bis-dihydrogenphosphate salt were stored in thedark at 25° C./60% relative humidity for 12 months (Trial 1, Batch 1)and 36 months (Trial 2, Batch 2), respectively. Both batch samples werealso stored in the dark under accelerated conditions at 40° C./75%relative humidity for 6 months. Samples were tested at appropriateintervals. The trials were performed in amber glass bottles equivalentto the full scale primary packaging unit.

The results demonstrate the long term stability of thebis-dihydrogenphosphate salt as shown in Table 3 (Trial 1) and Table 4(Trial 2) and FIGS. 4 to 6.

TABLE 3 Twelve month stability data for bis-dihydrogenphosphate salt(Trial 1, Batch 1) Assay (HPLC) Related Related Storage Water (% onSubstances Substances Storage Time Content Anhydrous (Major by HPLC)(Total by HPLC) Condition (Months) Appearance¹ (%) Basis) (Area %) (Area%) Initial 0 Complies 0.1 99.2 0.02 0.05 (RRT1.45) 25° C. 1 Complies 0.299.9 0.01 0.06 60% RH (RRT0.20, 0.25, 0.34, 0.53, 1.45, 1.95) 3 Complies0.1 99.7 0.02 0.07 (RRT0.35, 1.46) 6 Complies 0.1 99.9 0.02 0.06(RRT1.43, 2.02) 9 Complies 0.1 99.9 0.02 0.07 (RRT0.34) 12 Complies 0.199.6 0.02 0.07 (RRT1.46, 1.97) 40° C. 1 Complies 0.1 100.2 0.02 0.06 75%RH (RRT1.46) 3 Complies 0.1 99.7 0.03 0.10 (RRT1.45) 6 Complies 0.1 99.80.02 0.06 (RRT1.46) ¹Complies = Yellow free flowing powder. Packagingintegral.

TABLE 4 Thirty-six month stability data for bis-dihydrogenphosphate salt(Trial 2, Batch 2) Assay (HPLC) Related Related Storage Water (% onSubstances Substances Storage Time Content Anhydrous (Major by HPLC)(Total by HPLC) Condition (Months) Appearance¹ (%) Basis) (Area %) (Area%) Initial 0 Complies 0.3 99.8 0.02 (RRT1.45) 0.02 25° C. 1 Complies 0.299.6 0.03 (RRT1.45) 0.03 60% RH 3 Complies 0.1 99.9 0.02 (RRT1.46) 0.026 Complies 0.1 99.6 0.03 (RRT1.46) 0.03 9 Complies 0.2 99.8 0.02(RRT1.45) 0.02 12 Complies 0.3 100.0  0.02 (RRT 1.47) 0.02 18 Complies0.1 99.5 0.03 (RRT1.45) 0.03 24 Complies 0.2 99.4 0.03 (RRT1.44) 0.03 36Complies 0.0 99.9 0.03 (RRT1.45) 0.03 40° C. 1 Complies 0.3 100.4 0.03(RRT1.45) 0.03 75% RH 3 Complies 0.0 99.8 0.02 (RRT1.46) 0.02 6 Complies0.1 99.9 0.03 (RRT1.46) 0.03 ¹Complies = Yellow free flowing powder.Packaging integral.

Whilst the acid addition salts of the present invention are describedwith reference to a seemingly exact molar ratio of the anion to cation,it is understood that the acid addition salts of the present inventionmay comprise a mixture of the salt, free base and where appropriateexcess acid depending on the reaction conditions used to form the salt.For example, if less than 1 molar equivalent of sulfuric acid (forexample 0.7 eq) is reacted with the free base of formula I, the isolatedproduct of the reaction is likely to contain a certain proportion of thesalt of formula II, together with a certain proportion of the free baseof formula I. The mixture may, however, still exhibit many of theadvantageous properties of the salt formed using 1 molar equivalent ofthe acid. Likewise, if greater than 2 molar equivalents of phosphoricacid (for example 2.6 eq) is reacted with the free base of formula II,the isolated product is likely to contain a certain proportion of thesalt of formula III, together with a certain proportion of excessphosphoric acid. The mixture may, however, still exhibit many of theadvantageous properties of the salt so-formed using 2 molar equivalentsof the acid. It is understood that such mixtures fall within the scopeof the invention. Accordingly in a preferred embodiment of the inventionthere is provided a mixture comprising, more preferably consistingessentially of, the acid addition salt of the compound of formula I.

The present invention also provides a method of forming the salt offormula II, comprising contacting the free base of formula I withsulfuric acid. The present invention also provides a method of formingthe salt of formula III, comprising contacting the free base of formulaI with phosphoric acid. It is understood that in the formation of thesalts, it is possible that the final product will be contaminated withsalts of stoichiometry which is different to the stoichiometry of thesalts of formulae II and III. In some embodiments, the salt of formulaII is formed by contacting the free base with between 0.5 and 1.5 molarequivalents (preferably between 0.9 and 1.1 molar equivalents) ofsulfuric acid. In some embodiments, the salt of formula III is formed bycontacting the free base with between 1.5 and 2.5 molar equivalents(preferably between 1.8 and 2.2 molar equivalents) of phosphoric acid.In each case, it is preferable to use a reaction solvent or solventswhich allow complete dissolution of the free base andsulfuric/phosphoric acid, but which are poor solvents for the productsalt of formula II/III. Isopropanol is preferable for the formation ofthe bis-dihydrogenphosphate salt, whilst ethanol and less so methanolprovide examples of other alternatives. It is understood that bothphosphoric acid and sulfuric acid often contain some quantity of water.Judicious choice of the reaction solvent allows for the facileseparation of the salt from the mother liquor and is a technique wellknown in the art. Alternatively, it may be possible to avoid the use ofa solvent through the use of either neat sulfuric acid or neatphosphoric acid. In any event, it is understood that the skilled chemistwould readily modify the temperature of the reaction mixture duringand/or after reaction in order to optimise the formation of the saltand/or optimise the recovery of the salt from the reaction mixturefollowing salt formation. For example the free base and acid may beinitially heated to effect dissolution and/or facilitate thoroughmixing, before the reaction mixture is cooled to reduce the amount ofthe salt that remains in solution.

Whilst it is preferable that the acid addition salts of the presentinvention exist in essentially crystalline form, it is understood thatsuch compounds may exist in partially crystalline or even amorphous formdepending on, for example, the conditions under which the solid productforms. These forms are also contemplated by the present invention. It isfurther understood that certain crystalline compounds may exist indifferent crystalline forms—a phenomenon known as polymorphism. Theseindividual crystalline forms and mixtures thereof are also contemplatedby the present invention. It is understood that the skilled person wouldbe aware of a number of different parameters that may be varied toaffect the nature of the solid produced through the action of the acidon the free base of formula I.

The present invention also relates to solvates (including hydrates) ofthe acid addition salts of formulae II and III. Solvents used forcrystallisation may include alcohols (eg methanol, ethanol, i-PrOH),aldehydes, ketones (eg acetone), esters (eg ethyl acetate) and water,each of which may inevitably be to some degree embedded within the saltproducts. The amount and location of the solvent molecule(s) within thesalt product depend on such factors as crystallisation solvent(s), thetemperature under which crystallisation occurs, the pressure under whichcrystallisation occurs, the rate of crystallisation, inter andintra-molecular forces, storage and drying conditions followingisolation, etc. Preferably the solvent used to form the salt products ispharmaceutically acceptable.

The present invention also provides solid and liquid pharmaceuticalcompositions comprising one or both of the salts of the presentinvention, preferably in a therapeutically effective amount, togetherwith one or more pharmaceutically acceptable solid and/or liquidcarriers. As used herein, the term “pharmaceutically acceptable carrier”encompasses such terms as filler, diluent, excipient, additive orencapsulating substance, and may be safely used in topical, local orsystemic administration. The pharmaceutically active derivatives of thepresent invention can be readily formulated with pharmaceuticallyacceptable carriers well known in the art such as flavouring agents,solubilizers, lubricants, suspending agents, binders, preservatives,tablet disintegrating agents and encapsulating materials. Such carriersenable the compounds of the invention to be formulated in dosage formssuch as tablets, powders, cachets, lozenges, pills, capsules, liquids,gels, syrups, slurries, suspensions and the like. These carriers arewell known in the art and may include sugars, starches, cellulose andits derivatives, malt, gelatin, magnesium carbonate, magnesium stearate,talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginicacid, phosphate buffered solutions, emulsifiers, pectin, tragacanth, lowmelting wax, cocoa butter, isotonic saline and pyrogen-free water.

The surprising properties of the salts of the present invention makethem particularly suitable for use in solid pharmaceutical compositions.For example, their lack of hygroscopicity facilitates ease of handling(weighing, transportation, etc), whilst their stability facilitatesextended shelf-life following synthesis and isolation. In particular,pharmaceutical compositions for oral use can be obtained by combiningthe active compounds with solid excipients, optionally grinding theresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients include fillers such as sugars (including lactose,glucose, sucrose, mannitol or sorbitol) and cellulose preparations suchas maize starch, wheat starch, rice starch, potato starch, gelatin, gumtragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarboxymethylcellulose as well as polyvinylpyrrolidone (PVP). Ifdesired, disintegrating agents such as cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof (such as sodiumalginate) may be added. The invention also provides dragee corescomprising suitable coatings. For this purpose, concentrated sugarsolutions may be used, which may optionally contain gum arabic, talc,polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments may be added to the tablets ordragee coatings for identification or to characterize differentcombinations of active compound doses.

According to a preferred embodiment the pharmaceutical compositioncomprising the acid addition salt of the compound of formula I inaccordance with the invention is formulated for oral delivery.

Pharmaceuticals which may be used orally include push-fit capsules madeof gelatin, as well as soft, sealed capsules made of gelatin and aplasticiser, such as glycerol or sorbitol. The push-fit capsules cancontain the active ingredients in admixture with filler such as lactose,binders such as starches, and/or lubricants such as talc or magnesiumstearate and, optionally, stabilizers. In soft capsules, the activecompounds may be dissolved or suspended in suitable liquids, such asfatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added.

The comparatively high aqueous solubility of each of the salts of theinvention makes them suitable for use in pharmaceutical compositionsthat comprise polar (especially aqueous) solvent(s). In particular,liquid pharmaceutical compositions provided by the invention may containacceptable diluents and carriers which are familiar to those skilled inthe art and include, but are not restricted to, saline, sterile water,ethoxylated and nonethoxylated surfactants, poly(ethylene glycol),poly(propylene glycol), fatty alcohols, fatty acids, hydrocarbon oils(such as palm oil, coconut oil, and mineral oil), cocoa butter waxes,silicon oils, pH balancers, cellulose derivatives, emulsifying agentssuch as non-ionic organic and inorganic bases, synthetic gums, resins,preserving agents, wax esters, steroid alcohols, triglyceride esters,phospholipids such as lecithin and cephalin, polyhydric alcohol esters,fatty alcohol esters, hydrophilic lanolin derivatives, hydrophilicbeeswax derivatives and host molecules such as cyclodextrins (forexample α-, β- and/or γ-cyclodextrin) which may modify exposure of partsof the guest molecule (for example the salts of the invention) tosolvent.

The compositions of the present invention may also be administered tothe respiratory tract as a nasal or pulmonary inhalation aerosol orsolution for a nebuliser, or as a microfine powder (preferably withparticles of the order of 1 to 10 microns in size or less) forinsufflation, alone or in combination with an inert carrier such aslactose, or with other pharmaceutically acceptable excipients. Aerosolformulations include those in which the salt is provided in apressurized pack with a suitable propellant such as a chlorofluorocarbon(CFC) for examples dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. Theaerosol may conveniently also contain a surfactant such as lecithin. Thedose of the drug may be controlled by provision of a metered valve. Thesalt may also be provided in a pharmaceutical formulation which forms agel in the nasal cavity. The powder composition may be presented in unitdose form for example in capsules or cartridges of eg gelatin, orblister packs from which the powder may be administered by means of aninhaler. Accordingly, in yet another preferred embodiment, thepharmaceutical composition comprising the acid addition salt of thecompound of formula I in accordance with the invention is formulated forintranasal delivery.

The pharmaceutical compositions of the present invention may provide forthe sustained release of the active compound.

Preferably the pharmaceutical compositions of the present invention arepresent in unit dosage forms.

The pharmaceutical compositions of the present invention may alsocomprise one or more other therapeutic and/or prophylactic ingredients.The salts of this invention may be useful in combination with knownanti-viral or anti-retroviral agents or other pharmaceuticals used inthe treatment of viral infections. Representative examples of theseadditional pharmaceuticals include immunomodulators, immunostimulants,antibiotics and anti-inflammatory agents. Exemplary anti-viral agentsinclude zanamivir, rimantidine, amantidine, ribavirin, AZT, 3TC, (−)FTC, acyclovir, famciclovir, penciclovir, ddI, ddC, ganciclovir,saquanivir, loviride, other non-nucleotide reverse transcriptase (RT)inhibitors and protease inhibitors, antiviral and antireceptorantibodies and receptor analogues, such as ICAM-1. Exemplaryimmunomodulators and immunostimulants include various interleukins,cytokines and antibody preparations. Exemplary antibiotics includeantifungal agents and antibacterial agents. Exemplary anti-inflammatoryagents include glucocorticoids and non-steroidal anti-inflammatorycompounds.

As used herein, the term “effective amount” relates to an amount of oneor more active substances that is effective in producing a desiredoutcome in a subject in need of treatment or prophylaxis, either in asingle dose or as part of a series. Such an effect may be measured, forexample, through a decrease in the number of particles of a particularvirus or viruses, or by the relief of symptoms within the subject. Theeffective amount will vary depending upon the health and physicalcondition of the individual to be treated, the taxonomic group of thesubject to be treated, the formulation of the composition, theassessment of the medical situation, and other relevant factors. Thequantity of the pharmaceutically active compounds(s) to be administeredmay depend on the subject to be treated inclusive of the age, sex,weight and general health condition thereof. It is expected that theeffective amount will fall in a relatively broad range that can bedetermined through routine trials. In some instances, administration ofan “effective amount” of active may produce a 50% reduction in symptomsand/or number of relevant virus particles in the subject. The dose ofactive compounds administered to a subject should be sufficient toachieve a beneficial response in the subject over time such as areduction in, or relief from, symptoms of viral infection. In thisregard, precise amounts of the active compound(s) for administrationwill depend on the judgment of the practitioner. In determining theeffective amount of the active compound(s) to be administered, thephysician may evaluate the severity of the symptoms associated withviral infection and/or some other means of determining the extent of theviral infection. Formulations containing one hundred (100) milligrams ofactive ingredient or, more broadly, 0.1 to four hundred (400) milligramsper tablet, are accordingly suitable representative unit dosage forms.

As used herein, the term “subject” or “individual” or “patient”, may beused interchangeably, and refer to any subject, preferably a vertebratesubject, and even more preferably a mammalian subject, for whomtreatment or prevention is desired. Most preferably the subject is ahuman in need of treatment or prevention of a virus, preferably of thepicornaviridae family, especially HRV.

Accordingly in another aspect the present invention provides a method oftreating a picornavirus infection in a subject in need thereofcomprising administering a salt of formula II and/or formula III.

In another aspect the present invention provides a method of preventinga picornavirus infection in a subject in need thereof comprisingadministering a salt of formula II and/or formula III.

In another aspect the present invention provides the use of a salt offormula II and/or formula III in the manufacture of a medicament for thetreatment of a picornavirus infection in a subject in need thereof.

In another aspect the present invention provides the use of a salt offormula II and/or formula III in the manufacture of a medicament for theprevention of a picornavirus infection in a subject in need thereof.

The picornavirus infection may be caused by any virus of the familyPicornaviridae. Representative family members include humanrhinoviruses, polioviruses, enteroviruses including coxsackieviruses andechoviruses, hepatovirus, cardioviruses, apthovirus, hepatitis A andother picornaviruses not yet assigned to a particular genus, includingone or more of the serotypes of these viruses. Preferably the inventionis used in the prevention or treatment of infection caused by one ormore serotypes of rhinovirus.

The terms “treatment” and “treating” as used herein cover any treatmentof a condition or disease in a subject, preferably a mammal (non-humanmammals or humans), more preferably a human, and includes: (i)inhibiting the viral infection, i.e., arresting its proliferation; (ii)relieving the infection, i.e. causing a reduction in the severity of theinfection; or (iii) relieving the conditions caused by the infection,i.e. symptoms of the infection. The terms “prevention” and preventing”as used herein cover the prevention or prophylaxis of a condition ordisease in a subject, preferably a mammal (non-human mammals or humans),more preferably a human and includes preventing the viral infection fromoccurring in a subject which may be predisposed to infection but has notyet been diagnosed as being infected.

The invention will now be described with reference to some examples. Itis to be understood that the particularity of the following descriptionis not to supersede the generality of the preceding description of theinvention.

Example 16-{2-[1-(6-methyl-3-pyridazinyl)-4-piperidinyl]ethoxy}-3-ethoxy-1,2-benzisoxazolephosphate salt [formula III]

Free base of formula I (1 kg) was added to isopropanol (24 L). Thesuspension was heated to reflux, dissolving the base. An aqueoussolution of phosphoric acid (81% w/w, 0.63 kg, 2.1 eq) in isopropanol(2.3 L) was heated to 80° C., before being added rapidly, withagitation, to the refluxing solution of the free base. A precipitateformed immediately. The slurry was heated at between 78° C. and 82° C.for 5 minutes before being cooled to room temperature. The solid productwas washed with isopropanol (3×10 L) then acetone (3×10 L) before beingdried at 40° C. in vacuo to constant mass, affording the title compoundas a bright yellow crystalline solid (1.5 kg, 97%).

Example 26-{2-[1-(6-methyl-3-pyridazinyl)-4-piperidinyl]ethoxy}-3-ethoxy-1,2-benzisoxazolesulfate salt [formula II]

A solution of the free base of formula I (1 g) in tetrahydrofuran (52mL) was added to an aqueous solution of sulfuric acid (0.05 M; 52 mL; 1eq). The mixture turned immediately cloudy. The salt was isolated byslow removal of solvent according to the following pressure reductionprogram at room temperature:

Time (h) Pressure 00:00:00 600 mbar 00:30:00 600 mbar 01:30:00 500 mbar02:30:00 400 mbar 05:30:00 300 mbar 08:30:00 200 mbar 11:30:00 100 mbar14:30:00  1 mbar 18:00:00  1 mbar

Example 3

Pharmaceutical compositions comprising the salt of formula III wereprepared by filling brown size 0 hard gelatin capsules with the salt andmade to weight with anhydrous glucose. The compositions of the capsulesare summarised in Table 5.

TABLE 5 Components Qty/capsule (mg) Function salt of formula III 10 2550 100 200 Active drug substance Glucose (anhydrous) QS QS QS QS QSDiluent Brown size 0 hard  1  1  1  1  1 Capsule gelatin capsules QS = asufficient quantity to meet the pre-determined fill weight.

Example 4 X-Ray Crystallography

X-Ray crystal structures were obtained for the monosulfate andbis-dihydrogenphosphate salts after re-crystallisation from methanol.

(i) Monosulfate Salt (Molecular Formula [C₂₁H₂₈N₄O₃](SO₄))

Six different solvents (ethanol, methanol, acetone, tetrahydrofuran,acetonitrile and 2-propanol) were evaluated for the recrystallisation ofthe monosulfate salt. Methanol was selected as it showed the bestsolubility of the salt at elevated temperatures. 20 mg of themonosulfate salt was dissolved in warm methanol at 40° C. and thenslowly cooling to room temperature gave rise to the formation of yellowcrystals. A yellow crystal was mounted on a Stoe Mark II-Imaging PlateDiffractometer System (Stoe & Cie, 2002) equipped with agraphite-monochromator.

Data collection was performed at −100° C. using Mo-Kα radiation(λ=0.71073 Å). 180 exposures at 10 min per exposure were obtained at animage plate distance of 135 mm, φ=0° and) <ω<180° with the crystaloscillating through 1° in ω. The resolution was D_(min)-D_(max)0.82-24.00 Å.

The monosulfate salt crystallised in a centrosymmetric monoclinicalcell, space group P2₁/c. The structure of this compound was solved bydirected methods using the program SHELXS-97 (Sheldrick, G. M., ActaCryst. (2008) A64, 112-122) and refined by full matrix least squares onF (Spek, A. L. J. Appl. Cryst. (2003) 36, 7-13) with SHELXH-97. The N—Hhydrogen atoms were derived from Fourier difference maps and thentreated as riding atoms while the remaining hydrogen atoms were directlyincluded in calculated positions and treated as riding atoms usingSHELXL-97 default parameters. All non-hydrogen atoms were refinedanisotropically. No absorption correction was applied. The crystal wasonly weakly diffracting related to its size (0.50×0.45×0.03 mm). Thecrystals were obtained as very thin plates. The structure was refinedwith acceptable standard uncertainties in bond distances and angles.

X-Ray Crystal Data for Monosulfate Salt Crystal Data

Crystal shape: plate; Crystal colour: yellow; Crystal size:0.50×0.45×0.03 mm; Empirical formula: C₂₁H₂₈N₄O₇S; Formula weight:480.53; Crystal system: Monoclinic; Space group: P 21/c; Unit celldimensions: a=22.578(4) A alpha=90 deg.; b=13.175(2) A beta=94.746(15)deg.; c=7.4843(13) A gamma=90 deg.; Volume: 2218.7(7) Â3; Cellrefinement parameters: Reflections 1873; Angle range 2.38<theta<21.30;

Z is 4;

Density (calculated) 1.439 g/cm̂3; Radiation used MoK\a; Wavelength0.71073 A; Linear absorption coefficient 0.198 mm⁻¹; Temperature 173(2)° K

Data Collection Details

Diffractometer STOE IPDS 2; Scan method rotation method; Number ofReflections measured 12038; Number of Independent reflections 2889;Number of observed reflections 1060; Criterion forrecognizing >2sigma(I); R(int)=0.2485; Theta range for data collection1.79 to 22.50 deg; Index ranges 24<=h<=24, −14<=k<=14, −8<=l<=8; F(000)1016

Refinement Details

Refinement method Full-matrix least-squares on F²; Final R indices[I>2sigma(I)] R1=0.1022, wR2=0.2222; R indices (all data) R1=0.2264,wR2=0.2816; R1 [=SUM(∥Fo|−|Fc∥)/SUM|Fo|]; wR̂2{=[SUM(w(Fô2−Fĉ2)̂2)/SUM(wFô4)]̂½}; H-locating and refining Method constr;Number of reflections used 2889; Number of L.S. restraints 4; Number ofrefined Parameters 298; Goodness-of-fit on F̂2 0.892; S {=[SUMw(Fô2−Fĉ2)̂2]/(n−p)̂½} n=number of reflections; p=Parameters used; calcw=1/[\ŝ2̂(Fô2̂)+(0.1225P)̂2̂]; where P=(Fô2̂+2Fĉ2̂)/3; Maximum delta/sigma0.000; Maximum e-density 0.374 e.Â−3; Minimum e-density −0.538 e.Â−3

Computer Programs used

Data collection program STOE X-AREA; Cell refinement program STOEX-AREA; Data reduction program STOE X-RED; Structure Solving ProgramSHELXS-97 (Sheldrick, 1990); Structure Refinement Program SHELXL-97(Sheldrick, 1997)

(ii) Bis-Dihydrogenphosphate Salt Monohydrate (Molecular Formula{[C₂₁H₂₈N₄O₃](H₂PO₄)₂(H₂O)})

Six different solvents (ethanol, methanol, acetone, tetrahydrofuran,acetonitrile and 2-propanol) were evaluated for the recrystallisation ofthe bis-dihydrogenphosphate salt. Methanol was selected as it showed thebest solubility of the salt at elevated temperatures. 20 mg of themonosulfate salt was dissolved in warm methanol at 40° C. and thenslowly cooling to room temperature gave rise to the formation of yellowcrystals. A yellow crystal was mounted on a Stoe Mark II-Imaging PlateDiffractometer System (Stoe & Cie, 2002) equipped with agraphite-monochromator.

Data collection was performed at −100° C. using Mo-Kα radiation(λ=0.71073 Å). 180 exposures at 10 min per exposure were obtained at animage plate distance of 135 mm, φ=0° and <ω<180° with the crystaloscillating through 1° in ω. The resolution was D_(min)−D_(max)0.82−24.00 Å.

The bis-dihydrogenphosphate salt crystallised in a centrosymmetricmonoclinical cell, space group C2/c. The compound crystallized as adi-cation with two H₂PO₄ ⁻ anions and one strongly disordered watermolecule. A disorder was found for one of the two anions resulting inpartial occupations for atoms O9, O10, O11, O9a, O10a and O11a(occupation 0.5). No hydrogen atoms were found for the H₂PO₄ ⁻ anionsbut they are included in calculations. The disordered and partiallyoccupied water molecules were refined isotropically. No hydrogen atomswere found for the water molecules but they are included incalculations.

The structure of this compound was solved by directed methods using theprogram SHELXS-97 (Sheldrick, G. M., Acta Cryst. (2008) A64, 112-122)and refined by full matrix least squares on F (Spek, A. L. J. Appl.Cryst. (2003) 36, 7-13) with SHELXH-97. The N—H hydrogen atoms werederived from Fourier difference maps and then treated as riding atomswhile the remaining hydrogen atoms were directly included in calculatedpositions and treated as riding atoms using SHELXL-97 defaultparameters. All non-hydrogen atoms were refined anisotropically. Noabsorption correction was applied. The crystal was only weaklydiffracting related to its size (0.45×0.40×0.05 mm). The crystals wereobtained as very thin plates. The structure was refined with acceptablestandard uncertainties in bond distances and angles.

X-Ray Crystal Data for Bis-Dihydrogenphosphate Salt Crystal Data

Crystal shape: plate; Crystal colour: yellow; Crystal size:0.45×0.40×0.05 mm; Empirical formula: C₂₁H₃₄N₄O₁₂P; Formula weight:596.46; Crystal system: Monoclinic; Space group: C 2/c; Unit celldimensions: a=54.964(14) A alpha=90 deg.; b=12.692(2) A beta=91.931(18)deg.; c=7.8918(15) A gamma=90 deg.; Volume: 5502(2) Â3; Cell refinementparameters: Reflections. 4485; Angle range

1.48<theta<21.98;

Z is 8;

Density (calculated) 1.440 g/cm̂3; Radiation used MoK\a; Wavelength0.71073 A; Linear absorption coefficient 0.226 mm⁻¹; Temperature 173(2)K

Data Collection Details

Diffractometer STOE IPDS 2; Scan method rotation method; Number ofReflections measured 19426; Number of Independent reflections 3608;Number of observed reflections 1416; Criterion forrecognizing >2sigma(I); R(int)=0.2505; Theta range for data collection1.48 to 22.50 deg; Index ranges −58<=h<=58, −13<=k<=13, −8<=l<=8; F(000)2512

Refinement Details

Refinement method Full-matrix least-squares on F²; Final R indices[I>2sigma(I)] R1=0.0995, wR2=0.2330; R indices (all data) R1=0.2114,wR2=0.2823; R1 [=SUM(∥Fo|−|Fc∥)/SUM|Fo|]; wR̂2 {=[SUM(w(Fô2−Fĉ2)̂2)/SUM(wFô4)]̂½}; H-locating and refining Method constr;Number of reflections used 3608; Number of L.S. restraints 7; Number ofrefined Parameters 342; Goodness-of-fit on F̂2 0.928; S {=[SUMw(Fô2−Fĉ2)̂2]/(n−p)̂½} n=number of reflections; p=Parameters used; calcw=1/[\ŝ2̂(Fô2̂)+(0.1225P)̂2̂]; where P=(Fô2̂+2Fĉ2̂)/3; Maximum delta/sigma0.000; Maximum e-density 0.610 e.Â−3; Minimum e-density −0.417 e.Â−3

Computer Programs Used

Data collection program STOE X-AREA; Cell refinement program STOEX-AREA; Data reduction program STOE X-RED; Structure Solving ProgramSHELXS-97 (Sheldrick, 1990); Structure Refinement Program SHELXL-97(Sheldrick, 1997)

Example 5 Salt Characterisation Methods and Parameters

The salts of the invention were characterised using the followingmethods and parameters.

Parameters for Raman Measurements

Spectrometer: Bruker RFS 100/S Raman spectrometer

Excitation Laser power: 400 mW

Resolution: 2 cm⁻¹

No. of scan: 128

Acquisition range: 3300-0 cm⁻¹

Aperture: 5.0 mm

Type of vial: RPD 96 well plate format glass vial or RPD 96 well plateformat glass vial with FEP lining.

Sample position: optimized to best signal intensity

Spectrum pre-treatment: Linear baseline correction, normalisation.

Parameters for NIR Measurements

Spectrometer: Brucker Vector 22 NIR spectrometer

Resolution: 2 cm⁻¹

No. of scan: 64

Acquisition range: 833-2500 nm.

Aperture: 5.0 mm

Type of vial: RPD 96 well plate format glass vial

Spectrum pre-treatment: Linear baseline correction.

CCD-Picture Acquisition

Camera: Sony XCD-C710CR

Objective: Navitar Precision Eye (Body tube art. no. 1-61449 withattachments 1.33 X (art. no. 1-41448) and 0.5 X (art. no.; 1-60110).

X-Ray Powder Diffraction

Spectrometer: Stoe Stadip diffractometer

Detector/geometry: Position sensitive detector/Debye-Scherrer geometry.

X-Ray source: Cu K alpha; Measurement mode: Transmission in 0.5 mmcapillary

Elemental Analysis

The following methods were used for the determination of the elementalcomposition:

C, H, N: Thermal decomposition and quantitative determination of theflue gas stream (Instrument Leco-900)

Cl: Ion chromatographic determination after Schoniger digestion.

S: Thermal decomposition and quantitative determination of the flue gasstream using an IR detector (Instrument LECO-CHN-932)

P: Photometric determination after microwave digestion.

DSC Analysis

The DSC analyses were performed on a Mettler Toledo differentialscanning calorimeter with a heating rate of 5° C. per minute.

Solubility Determination

Approximately 10 mg of the acid addition salt were equilibrated for 24hours at 25° C. with deionised water. The suspension was filtered, thecontent of the free base was determined by HPLC and the pH of thesaturated solutions was measured.

HPLC Analysis

A test solution of the acid addition salt was prepared by dissolving10.0 mg of the salt in acetonitrile:ethanol=1:1 and diluted to aconcentration of 1.0 mg/mL.

Chromatograph: TSP- Detector Spectra-Physics SP877 XR Layer thickness: 8mm. Detection wavelength: 230 nm (UV detection) Output voltage: 1 VColumn: Nucleosil C18 Particle size: 5 μm Column length: 250 mm Columni.d.: 4.0 mm Column temperature: room temperature Size of sample: 10 μlof reference/test solution Flow rate: 1.0 ml/minute Gradient program 1:Time 0.1% aqueous Acetonitrile [minutes] phosphoric acid [%] [%] 0 70 3035 1 99 36 70 30 41 70 30 Evaluation: Calculation of area-%

Exemplary HPLC Assay Data for Bis-Dihydrogenphosphate SaltChromatographic Conditions

Equipment: HPLC system fitted with a UV Detector Column: WatersSymmetry, C8, 5 μm, 150 mm × 3.9 mm Column temperature: 25° C. Detectorwavelength: 265 nm Flow rate: 1.0 mL/min Injection volume: 10 μL Mobilephase: 0.1M KH₂PO₄ (pH 3.0):Methanol (45:55) Run time: 30 minutesDiluent: Mobile phase Needle wash: Methanol (100%)

Under these conditions, the retention time for thebis-dihydrogenphosphate salt is approximately 9-11 mins.

An exemplary HPLC chromatogram is shown in FIG. 9 having the followingpeak results:

Name Int Type RT Height Area % Area BB 2.623 180 1311 0.02 BB 5.489 1351697 0.02 Bis- BB 10.641 366923 7581975 99.93 dihydrogen- phosphate saltbb 15.555 113 2575 0.03

Stability

Approximately 30 mg of the salt was stored in a 96 well glass vial forfour weeks under the following conditions:

-   -   25° C./dry    -   25° C./75% relative stability    -   40° C./dry    -   40° C./75% relative stability

During storage of the test samples, NIR and Raman spectra were acquired(non-invasively) at pre-selected time points. The Raman and NIR spectradata sets were analysed with respect to hygroscopicity (NIR),morphological (NIR and Raman) and chemical changes of the testsubstance. HPLC analysis of the test substances was performed at the endof the storage period.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integers or steps.

The reference in this specification to any prior publication (orinformation derived from it), or to any matter which is known, is not,and should not be taken as an acknowledgment or admission or any form ofsuggestion that that prior publication (or information derived from it)or know matter forms part of the common general knowledge in the fieldof endeavour to which this specification relates.

1-21. (canceled)
 22. A method of treating a picornavirus infection in asubject in need thereof, comprising the step of administering to thesubject an effective amount of a crystalline form of6-{2-[1-(6-methyl-3-pyridazinyl)-4-piperidinyl]ethoxyl-3-ethoxy-1,2-benzisoxazolebis-dihydrogenphosphate salt (1:2), wherein the crystalline form has anX-Ray Powder Diffraction pattern with 20 peaks (±0.2°) at 3.3°, 6.7°,12.8°, 13.3°, 14.1°, 19.2°, 20.0°, 21.1° and 22.4°.
 23. The method ofclaim 22 wherein the picornavirus infection is human rhinovirus (HRV).24. The method of claim 22 wherein the crystalline form is administeredas a pharmaceutical composition, wherein the pharmaceutical compositioncomprises the crystalline form and a pharmaceutically acceptablecarrier.
 25. A method of preventing a picornavirus infection in asubject in need thereof, comprising the step of administering to thesubject an effective amount of a crystalline form of6-{2-[1-(6-methyl-3-pyridazinyl)-4-piperidinyl]ethoxy}-3-ethoxy-1,2-benzisoxazolebis-dihydrogenphosphate salt (1:2), wherein the crystalline form has anX-Ray Powder Diffraction pattern with 20 peaks (±0.2°) at 3.3°, 6.7°,12.8°, 13.3°, 14.1°, 19.2°, 20.0°, 21.1° and 22.4°.
 26. The method ofclaim 25 wherein the picornavirus infection is human rhinovirus (HRV).27. The method of claim 25 wherein the crystalline form is administeredas a pharmaceutical composition, wherein the pharmaceutical compositioncomprises the crystalline form and a pharmaceutically acceptablecarrier.
 28. The method of claim 27 wherein the pharmaceuticalcomposition is formulated for oral delivery.
 29. The method of claim 27wherein the pharmaceutical composition is formulated for intranasaldelivery.
 30. The method of claim 27 wherein the pharmaceuticalcomposition is in a unit dosage form.
 31. The method of claim 27 whereinthe pharmaceutical composition further comprises glucose.
 32. The methodof claim 27 wherein the pharmaceutical composition provides forsustained release of the crystalline form.
 33. The method of claim 27wherein the pharmaceutical composition is in the form of a tablet,powder, cachet, lozenge, pill or capsule.
 34. The method of claim 27wherein the pharmaceutical composition is in the form of a liquid, gel,syrup, slurry or suspension.
 35. The method of claim 27 wherein thepharmaceutical composition is in the form of an inhalation aerosol,nebulizer solution, or microfine insufflation powder.
 36. The method ofclaim 27 wherein the pharmaceutical composition further comprises ananti-viral agent, an anti-retroviral agent, and/or a pharmaceutical fortreatment of viral infections.
 37. The method of claim 22 furthercomprising the step of administering to the subject an anti-viral agent,an anti-retroviral agent, and/or a pharmaceutical for treatment of viralinfections.
 38. The method of claim 25 further comprising the step ofadministering to the subject an anti-viral agent, an anti-retroviralagent, and/or a pharmaceutical for treatment of viral infections.